The emerging discipline of cryo-electron tomography provides unique opportunities to determine 3-dimensional cellular architectures in their native conditions at resolutions one to two orders of magnitude higher than what is currently achieved using light microscopy. It bridges the critical gap between high resolution structure determination of protein complexes by NMR or X-ray crystallographic techniques and single-particle living cell imaging by light microscopy using fluorescent probes. Advances made in recent years to automate data acquisition using modern computerized microscopes have enabled this technology to image complex assemblies within the native cells and to determine the 3D architecture of cells in their native states. Despite the acknowledged potential of this methodology, one major limitation, specimen thickness, has hindered its broader application in cellular and structural biology. Until now, most of the cryo-electron tomographic studies have been confined to bacterial cells and viruses or thin areas at the leading edge of cells, where useful information can be recovered from cellular tomograms. Extension of this technology to large cells, mammalian cells in particular (>1 um), and even high-pressure frozen tissues requires thinning of the cryo-specimen to less than a half micron in thickness. We will explore using a focused ion beam (FIB) to thin the frozen-hydrated specimen to a degree suitable for cryo-electron tomography. This method could eliminate common difficulties and artifacts associated with cryo-ultramicrotomy and significantly advance cryo-electron microscopy (cryoEM), particularly cryo-electron tomography, in studying the 3D architectures of cellular assemblies, organelles, cells and tissues in their native state. To this end, we propose the following specific aims: Aim 1: Develop a cryo-stage for thinning frozen-hydrated specimens within the FIB instrument;Aim 2: Characterize the cryo-FIB milling process with plunge-frozen bacterial cells and large mammalian cells;Aim 3: Analyze the 3D architectures of cryo-FIB thinned bacterial and mammalian cells using cryo-electron tomography. The development and implementation of this methodology to thin biological specimens preserved in a frozen-hydrated native state will overcome the major limitation on using such specimens in cryoEM and permit use of cryo-electron tomography as a standard technology for high-resolution 3D imaging of native cells and tissues. The proposed methodology could then be applied to a wide range of biomedical and translational research initiatives, such as spatial localization of tagged therapeutic drugs within the cancer cells in its native state and analysis of structural and morphological changes of cancer cells upon various drug treatments. Public Health Relevance Statement: The development and implementation of this methodology will overcome the major limitation in imaging of biological specimens preserved in a frozen-hydrated native state using cryo-electron microscopy and permit use of cryo-electron tomography as a standard technology for 3D imaging of native cells and tissues at resolution 10 to 100 times higher than that obtained with confocal microscopy. The proposed methodology could then be applied to a wide range of biomedical and translational research initiatives, such as spatial localization of tagged therapeutic drugs within the cancer cells in their native state and analysis of structural changes of cancer cells upon various drug treatments;study the intricate interplays between HIV and its host cellular components that are essential for HIV pathogenesis and providing structural information on how virus utilizes the host machinery both to promote its replication and, at the same time, to subvert and evade the antiviral responses of the cell.